1. Field of the Invention
The present invention relates to detection of the existence of a specified sequence of oligonucleotide which is an interesting analyte existing in a nucleic acid sample. The present invention is effective particularly in detection of pathogenic microorganisms such as infectious bacteria and is used for diagnoses and screenings of infectious diseases.
2. Description of the Related Art
Detection and identification of pathogenic microorganisms are indispensable for diagnoses of infectious diseases. Therefore, the antibody titer of the serum of a patient is measured for the specific antigen of a specified pathogenic microorganism. Such a measurement, however, is a mere detection of the shadow of the infectious diseases and is not confirmation of their essence. Detection and identification by cultivation of a microorganism responsible for an infectious disease has been disadvantageous in that it requires a long period of time and hence cannot be employed for practical diagnosis and treatment of the disease.
Investigation of the existence of a specified base sequence in a nucleic acid sample permits specification of a microorganism responsible for an infectious disease and diagnosis of the infectious disease before crisis.
For example, there is a method using a DNA probe. In this method, a single-stranded DNA having a base sequence complementary to the base sequence of a nucleic acid to be detected (the single-stranded DNA is referred to as a "DNA probe") is utilized as a specifically reactive reagent. The existence of objective pathogenic bacteria can be judged by investigating the existence, in a sample, of a base sequence complementary to the base sequence of the DNA probe (E. M. Southern, J. Mol. Biol., 98, 503-517 (1975) and J. Meinkoth, Anal. Biochem., 138, 267-284 (1984)).
As an example of the method using the DNA probe, dot hybridization comprises attaching a single-stranded DNA (an SS-DNA) obtained by denaturation of a sample to a solid phase, allowing a radioisotope-labeled SS-DNA to act on the solid phase to form a hybrid between the labeled SS-DNA and the SS-DNA attached to the solid phase, removing the unreacted labeled SS-DNA, and measuring radiation emitted from the solid phase.
As a modification of the above method, there is sandwich hybridization. This method makes it possible to reduce the background due to adsorption and hence is effective particularly when an impure sample is used. In this method, at least two DNA fragments derived from a target nucleic acid to be recognized are used. One of the DNA fragments is attached to a solid phase and used as a capturing reagent. The other fragment is labeled as a reagent for detection and added to a hybridizing solution together with a solubilized reference sample. When a base sequence homologous with both reagents exists in the reference sample, the sequence ought to be hybridized with both the capturing reagent and the reagent for detection. Whether the sequence has been hybridized or not can be known through labeling of the solid phase.
The above two methods are disadvantageous particularly when the amount of a nucleic acid to be detected is small. In addition, they have been disadvantageous from the viewpoint of labor and time required for operations because a large number of steps are necessary for measurement and a long time is required, particularly for immobilizing a sample.
A method capable of solving the above problems has been proposed, for example, in Japanese Patent Application Kokoku No. 3-78120. This method uses a restriction enzyme. It comprises bringing, in a solution, a single-stranded polynucleotide to be measured into contact with a solid phase to which a labeling substance, a single-stranded polynucleotide to be measured and a double-stranded polynucleotide have been attached, thereby forming a double-stranded polynucleotide; allowing a restriction enzyme to cleave the formed double-stranded polynucleotide and measuring the labeling substance in the solution or solid phase.
U.S. Pat. No. 5,118,605 discloses a method using a restriction enzyme or a reagent having an optional cleavage position introduced thereinto.
In the measurement of nucleic acids by the use of DNA probes, reagents containing different respective DNA probes should be prepared for different analytes to be measured (i.e. sequences of oligonucleotide which are interesting analytes existing in nucleic acid samples). Since the development of the DNA probes requires a great deal of labor, the development of the reagents for the measurement tends to be retarded. This has been a cause of the retardation of generalization of a method for detecting infectious bacteria by investigating the existence of a specified sequence of oligonucleotide, which is an interesting analyte existing in a nucleic acid sample.
In reducing measurement errors due to nonspecific adsorption or the like, a method comprising recognition of a double strand formed by combination and selective cleavage is also effective. However, analysis using different DNA probes for different analytes to be measured requires recognition of the double strand by different restriction enzymes for the different analytes to be measured and selective cleavage of the double strand. Therefore, for developing reagents for measurement, it has been necessary to compose a measuring kit by preparing the different restriction enzymes for the different analytes to be measured. Since a great deal of labor is required for developing a restriction enzyme capable of recognizing a special sequence of oligonucleotide (a specified base sequence) and cleaving the same selectively, the development of the reagents for measurement tends to be retarded and moreover only very expensive restriction enzymes have been developed. This has been another cause of the retardation of generalization of a method for detecting infectious bacteria by investigating the existence of a specified sequence of oligonucleotide which is an interesting analyte.
Thus, in the conventional methods, reagents quite separately developed should be prepared for analytes to be measured, respectively.